Determination of bacteria



US. Cl. 195-103.5

3,5325% Patented Oct. 6, 1970 3,532,603 DETERMTNATION OF BACTERIA Ronald Freake, Dandenong, Victoria, Australia, assignor to Castro! Limited, London, England, a British com- P y No Drawing. Filed Aug. 14, 1967, Ser. No. 660,207 Claims priority, application Australia, Aug. 15, 1966, 9,674/66 Int. Cl. C12k 1/02 15 Claims ABSTRACT OF THE DISCLOSURE A field method of determining the concentration of viable bacteria in a medium by means of an absorbent strip or other indicator device which carries methylene blue or other substance which undergoes a colour change in the presence of bacteria. The methylene blue or other substance is present in different concentrations in different parts of the device and the concentration of bacteria in the medium is indicated by the extent to which the colour change proceeds in various parts of the device after the device has been immersed in the medium to be tested.

This invention relates to a method of determining the concentration of bacteria present in a medium. It also relates to an indicator device which may be used in such a method and to methods of preparing said device.

The invention is particularly concerned with bacteria in water soluble oils, particularly cutting oils. However, it also has application in determining the concentration of bacteria in other media, such as jet engine fuels and other petroleum products. Other applications are in connection with the determination of the concentration of bacteria in dairy products and for medical diagnostic use such as in the detection of urinary tract infections.

The invention will be described hereinatfer in relation to soluble oils. However, it should be borne in mind that the invention is applicable to other oils and to other media.

Soluble oils are complex mixtures of oil and other materials which, when mixed with water, form stable emulsions. They normally comprise emulsifiable petroleum oils or emulsifiable mixtures of petroleum and fatty oils. The emulsifying agents are generally soaps of petroleum sulphonates, resin, tall oil or fatty acids, while synthetic emulsifiers may also be employed.

Soluble oil emulsions are used in industry as c'oolants and as lubricants for cutting, drilling and grinding metal. They serve to reduce friction, dissipate heat and prolong the life of the cutting tools as well as improving the surface finish of the components being worked. They may also prevent warping of the machined metal and leave a rust-preventing film of oil on the finished work.

Many bacteria grow in soluble oil emulsions and cause such undesirable conditions as foul odours and lowered pH with subsequent breaking of the emulsion. The bacteria can also cause infection, for example, dermatitis type infections and represent an industrial hazard particularly when hygiene is not correctly followed.

One of the major difficulties in dealing with bacteria in media such as soluble oils has been the lack of a field method for determining the concentration of bacteria present in the medium and accordingly, the object of this invention is to provide a convenient and simple method for determining the presence and concentration of bacteria in such media. The invention also aims to provide a relatively cheap and convenient test indicator use in the aforesaid method and a process for making such test indicator.

According to this invention, we provide a method of determining the concentration of viable bacteria in a medium in accordance with which an indicator device is immersed in, or otherwise brought into contact with the medium to be tested, the indicator device having thereon or therein a substance capable of undergoing a colour change in the presence of bacteria such that the concentration viable bacteria present in the medium is indicated by the colour change occurring in the said substance.

This invention also provides a means for determining the concentration of viable bacteria in a medium, said eans comprising an indicator device having thereon or therein a substance capable of undergoing a colour change in the presence of bacteria such that, when the device is immersed in, or otherwise brought into contact with, a medium to be tested, the concentration of bacteria present in the medium is indicated by the colour change occurring in the said substance.

Further, this invention provides a method of making a test indicator for determining the concentration of viable bacteria in a medium which method comprises the steps of impregnating various areas of a piece of absorbent material with solutions of methylene blue and an additive capable of retaining the methylene blue in the material in use, and cutting the impregnated piece of absorbent material into strips, the solutions containing methylene blue in various concentrations to provide areas of methylene blue of progressively increasing concentration along the length of the strips.

The substance may be present in different concentrations on or in different parts of the indicator device so that the concentration of viable bacteria present in the medium is indicated by the extent of the colour change which occurs in the various parts of the indicator device.

The said substance may be one which undergoes a colour change as a result of removal of oxygen from the medium by reductase formed by bacteria. The pre ferred substance of this type is methylene blue. Nevertheless, the invention is not limited to the use of methylene blue as other compounds capable of detecting bacterial metabolism may be utilized. If desired the test indicator may carry two' or more such substances.

The test indicator may be made from absorbent material such as filter paper.

Preferably, the said substance is incorporated in a composition which contains an additive which is capable of retaining the said substance on or in the test indicator in use. A suitable additive is albumin. Alternatively, other materials such as alginates may be used for this purpose.

Sodium alginate is preferred because of its relatively high solubility.

In a typical procedure, methylene blue and albumin are applied to filter paper and the albumin subsequently denatured by drying at 65 C. for 30 minutes. The methylene blue may be applied in w/v concentrations of from 1/ 8,000 to 1/40,000 parts of diluent. Conveniently, the methylene blue is applied to various areas on the material in concentrations of 1/ 8,000; 1/ 16,000; 1/24,000; 1/32,000 and 1/40,000.

To aid in the further understanding of this invention, one method of preparing a test indicator in accordance with this invention, and its method of use, will now be described.

According to this procedure a piece of absorbent material such as filter paper is marked off into sections by parallel lines and every alternate section impregnated with a solution of methylene blue and albumin, the concentration of methylene blue progressively increasing in the different sections. Solutions of methylene blue having concentrations (w/v) of 18,000; 1/l6,000; l/24,000;

l/32,000 and 1/40 .000 are used for impregnating the ab sorbent material in this manner.

After being impregnated with the various solutions, the paper is dried for 30 minutes at 65 C. This treatment, as well as drying the paper, fixes the methylene blue by rendering the albumin insoluble in water. The methylene blue is thus prevented from running into the medium when it is being tested in the manner hereinafter explained.

After being dried, the piece of filter paper or other absorbent material is provided with a backing of stiller material and cut into strips at right angles to the lines separating the sections impregnated with the various solutions. In this way, elongated rectangular strips are pro duced such that each strip is provided along its length With alternate coloured and uncoloured square areas, the Coloured areas becoming progressively darker along the length of the strip.

Before being put into use, the strips are calibrated by being immersed for a standard time in various media containing known concentrations of viable bacteria. By observing which areas are thus decolorized, it is possible to correlate the extent of decolorization along the strip with the concentration of viable bacteria present in a medium.

In use, therefore, it is only necessary to place a strip in a medium for the standard time and then observe the extent of decolorization. A quantitative indication of the concentration of viable bacteria present in the medium can then be read off by comparison with a colour standard prepared on the basis of the calibration tests. It has been found in practice that an immersion time of between 10 and 20 minutes is satisfactory, but this period could be varied according to circumstances.

The alternate white areas on the strips are of assistance in determining which areas have been decolorized or partly decolorized as a white standard helps to improve the judgment particularly if the medium imparts a colour to the strips.

To faciltiate reading the results of a test, a sign such as the word CONTAMINATED or any other suitable mark, may be printed or otherwise applied to the coloured areas of the strip in a colour substantially matching the colour of the lightest coloured area. The sign is made in a fast ink or other colouring material not affected by exposure to the medium. In this case, the darkest area to be decolorized for the purpose of the test will be indicated by the darkest area in which the sign becomes visible on or after exposure to the medium.

The invention is illustrated by the following nonlimitative examples. In each case, freeze dried cultures of organisms obtained from contaminated coolants and turbine oils were rehydrated with peptone water and incubated for 6 hours at 37 C. The quantities of viable bacteria present in the various cultures were then determined by the method of the invention and the results Were compared with counts taken according to the well known Miles and Misra method (see J. Hyg. 385732, 1938).

For carrying out the method of the present invention, use was made of strips of No. l filter paper each divided along its length into ten square areas and treated such that alternate areas were impregnated with a solution of methylene blue and albumin. The concentration of the methylene blue progressively increased along the length of the strip such that in the first coloured area, the impregnant was an aqueous solution containing methylene blue in a concentration (w./v.) of l/40,000, while solutions containing concentrations of l/32,000; l/24,000; l/l6,000; and 1/8,000 were used for the other coloured areas.

In carrying out the tests, the peptone water cultures were divided into 2 aliquots. in one of which the concentration of viable bacteria was determined by the method of the invention and in the other of which the concentra- TABLE 1 Esch. coli Method of Miles and Misra 1 Method of invention 2 9x10 1 3X10 1 4x10 1 3x10 2 2X 10 3 5x10 2 2X10 4 4 1O 4 2X 10 3 2 10 4 4X 10 1 3 10 1 6x10 2 7 10 2 2X10 1 4X 10 5 2X10 1 5 10 3 4X 10 4 2 10 1 7X10 2 3 10 3 2X10 4 4x10 2 3x10 2 Number of viable organisms per ml. F Number of squares decolorized commencing from square with lowest concentrations of methylene blue.

TABLE 2 Staphylococcus Method of Miles and Misra 1 Method of invention 2 4x 10 4 6 x 10 5 4x 10 4 3 x 10 4 4 x 10 3 3 X 10 1 4x 10 3 8 X 10 4 8 X 10 5 4x 10 2 3 x 10 1 4x 10 1 6 X 10 5 4 X 10 3 3 X 10 3 4 X 10 4 5 X 10 3 1 Number of viable organisms per m1.

2 See footnote -2 in Table 1.

TABLE 3 Bactcraides Sp.

Method of Miles and Misra 1 Method of invention 2 4x10 2 3X10 4 4X10 2 3x10 4 4X10 2 4x10 3 3x10 2 1 Number of viable organisms per ml. 2 See footnote 2 in Table 1.

TABLE 4 Pseudomonas Sp.

Method of Miles and Misra 1 Method of invention 2 2X10 1 3x10 1 6x10 2 4x10 2 3 x10 2 4 10 3 3X10 4 4X10 1 5X 10 4 4 10 5 3X 10 2 2X10 1 2X 10 2 4x10 3 2 10 3 4X10 2 2x10 2 4x10 2 5 10 4 3X10 3 2X10 3 4X10 2 2X 1 1 4x10 2 3X 10 3 4X 10 3 1 Number of viable organisms per ml.

See footnote 2 in Table 1,

1: Second square partially decolorized.

' Fourth square partially deeolorized.

TABLE 5 Proteus Sp.

Method of Miles and Misra 1 Method of invention 2 3x10 3 4X10 4 4x10 5 3X 4 4x10 2 5x10 4 3 X 10 4 2X10 4 6X10 3 3 6x10 3 4 x10 4 3 2x10 2 4x10 2 8 10 3 9 10' 4 2x10 2 4X10 2 Number of viable organisms per ml. See footnote 2 in Table 1.

3 Fourth square partially decolorized. 4 Fourth square partially decolorized.

TABLE 6 Streptococci Method of Miles and Misra 1 Method of invention 2 4x10 4 3x10 4 2x10 1 6x10 1 4 10 1 5x10 2 6 1O 1 6 10 2 7x10 2 6 TABLE 6-Continued Method of Miles and Misra 1 Method of invention 2 Number of viable organisms per ml. See footnote 2 in Table 1.

3 Second square partially decolorized.

4 Third square partially decolorized.

TABLE 7 Mixed cultures Method of Miles and Misra 1 Method of invention 2 4x10 2 6 l0 3 3 10 1 2x10 2 2 10 2 4X10 3 3x10 1 Number of viable organisms per ml.

2 See footnote 2 in Table 1.

From the above it will be apparent that a correlation exists between the number of squares decolorized and the concentration of viable bacteria in the medium. This relationship is as follows:

Number of Squares Order of conccentration of viable Decolorized bacteria (approx. number per ml.)

4 10 -10 S 10 and above.

In the case of non-aqueous media such as engine fuels, a measured quantity of the material to be tested is mixed with an equal quantity of Ringers solution, a suitable method of mixing being by means of a Vortox J R mixing machine for 3 minutes. The layers are then allowed to separate and the aqueous layer tested by means of coloured strips in the manner described above.

As, however, not all the bacteria will pass into the aqueous phase, it is necessary to apply a correction factor to the results of the test to obtain a true indication of the concentration of viable bacteria in the medium. This factor can be ascertained for any particular type of medium by passing a measured quantity of the medium through a Millipore filter to extract the bacteria, and plate counts are then made on the extracted bacteria. Millipore filters are marketed by the Millipore Filter Corporation of Bedford, Mass, United States of America and the plate counts may be carried out by the method described by Mackie and McCartney in Handbook of Practical Bacteriology (9th edition) as modified in the Technical Data Manuals ADM 10 and ADM 30 published by the Millipore Filter Corporation. Alternatively, the medium may be subjected to examination on a Coulter particle counter or similar device in order to ascertain the total number of bacteria present in the sample. This method is, however, not as reliable as a plate count as the counter does not discriminate between viable bacteria and other particles in the medium.

Once the correction factor has been established for any particular type of non-aqueous medium, the method of the invention can be used in the same Way as for aqueous media.

The invention thus provides a relatively simple, convenient, quick and economical field method for determining the concentration of bacteria present in a medium. The problem of bacterial contamination of soluble oils, engine fuels and other media is now becoming widely recognized and the present invention will materially assist in dealing with this problem. It will also be apparent that the invention may be used in relation to contamination resulting from a broad spectrum of bacterial types.

I claim:

1. A method of determining the concentration of via'ble bacteria in a medium which comprises contacting an indication device with, the medium to be tested, said indicator device having thereon or therein a substance that undergoes a color change due to the action of bacteria such that the concentration of viable bacteria present in the medium is indicated by the color change of the said substance, said substance being present in different concentrations at different discrete parts of the indicator device and monitoring the extent of the color change which occurs in the different discrete parts of the indicator device to indicate the concentration of viable bacteria present in the medium.

2. A method as in claim 1, in which the said substance is incorporated in a composition which contains an additive capable of retaining the said substance on or in the test indicator in use.

3. A method as in claim 1, in which the said substance is methylene blue.

4. A method as in claim 3, in which the said additive is albumin.

5. A method as in claim 1 in which said substance is methylene blue and is incorporated in a composition containing albumin and the test indicator is immersed in the medium for a period of between 10 and 20 minutes.

6. A method as in claim 1, in which the extent of the color change is compared with a calibrated color standard.

7. Means for determining the concentration of viable bacteria in a medium, said means comprising an indicator device having thereon or therein a substance that undergoes a color change due to the action of bacteria such that, when the device is contacted with, the medium to be tested, the concentration of bacteria present in the medium is indicated by the color change occurring in the said substance, said indicator device having discrete portions and the said substance being present in different concentrations at different ones of said discrete portions so that the extent of the color change which occurs at the various discrete portions indicates concentration of viable bacteria present in the medium.

8. Means for determining the concentration of viable bacteria in a medium as in claim 7, in which the said substance is incorporated in a composition which contains an additive capable of retaining the said substance on or in the test indicator in use.

9. Means for determining the concentration of viable bacteria in a medium as in claim 7, in which the said substance is methylene blue.

10. Means for determining the concentration of viable bacteria in a medium as in claim 8, in which the said additive is albumin.

11. Means for determining the concentration of viable bacteria in a medium as in claim 8, in which the said additive is an alginate.

12. Means for determining the concentration of viable bacteria in a medium as in claim 7 in which the indicator device is formed of absorbent material.

13. Means for determining the concentration of viable "bacteria in a medium as in claim 7 in which the indicator device comprises a strip divided along its length into a series of discrete areas which carry the said substance in progressively increasing concentrations along the length of the strip.

14. Means for determining the concentration of viable bacteria in a medium as in claim 13, in which the discrete areas carrying the said substance in progressively increasing concentrations are separated by areas not carrying the said substance.

15. Means for determining the concentration of viable bacteria in a medium as in claim 10, in which the albumin has been denatured by means of a drying treatment.

References Cited UNITED STATES PATENTS 2,904,474 9/1959 Forg 103.5

3,066,081 11/1961 Rorem et al. 195-103.5

3,206,3' 17 9/1965 Golber 195103.5

ALVIN E. TANENHOLTZ, Primary Examiner 

